Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish

Abstract

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

Figure 1

GFP fluorescence locating and quantifying ERE activation by BPA and MBP in the heart in in embryo-larval zebrafish at 5 dpf: (a) Solvent control; (b) 1000 μg/L BPA; (c) 25 μg/L MBP; (d) Close up of the region of interest showing the heart (encircled). Fluorescence indicating Estrogen Response Element (ERE) activation was concentrated in the Atrio-Ventricular (AV) and Ventricular-Bulbous (VB) valves.

Figure 2

Images of atrio-ventricular (AV) valve leaflets from high-level BPA and MBP exposure treatments versus solvent controls at 15 days post fertilization (dpf) Bright field images a-c are shown at ×100 magnification: AV valve leaflets were bent in the high-level MBP exposure. TEM images d-l are shown at ×3000, ×6000, and ×20 000 magnification: The extra-cellular matrix between the bilayer of valvular cells was narrower and lacked collagen in the high-level BPA and MBP exposure treatments compared to solvent controls (qualitative assessment). Annotations: A = atrium, V = ventricle, E = erythrocytes, Va = valve leaflet, IS = interstitial space, CF = collagen fibers, N = nucleus, arrow heads indicate bent AV valve leaflet. Scale bar (bottom left in each image): a–c = 10 μm; d–f = 5 μm; g–i = 2 μm; j–l = 1 μm.

Figure 3

Effects of BPA and MBP exposure on cardiovascular function in zebrafish larvae at 15 days post fertilization (dpf) Hatched bar charts (A-B) represent BPA, solid bar charts (C–D) represent MBP. Data represent six individual fish taken randomly from each of six separate aquaria (n = 6 experimental replicates) per exposure treatment. Bar heights represent means, error bars represent 95% confidence intervals. Significant differences from 0 μg/L solvent control (p > 0.05) are highlighted with an asterisk.

Figure 4

Gene set enrichment for KEGG pathways in heart tissues from 5 and 15 day old larval zebrafish in BPA and MBP exposure treatments (versus solvent controls). Sequence data were generated from hearts pooled from ∼30 individuals from each of four separate aquaria (nominally n = 4 experimental replicates) per exposure treatment. Enriched pathways were identified using Enrichr and referenced to the KEGG database (2016). Pathways highlighted in red boxes are calcific aortic valve disease (CAVD) biomarkers. Also see enriched Reactome pathways (in SI Figure S8).